I have tried to purify Pichia secretory protein of 80kDa. Initially precipitated the soup protein using ammonium sulphate precipitation. Then resuspended the precipitation in 1x PBS ( 20mM potassium phosphate, pH 7.4, 300mM NaCl and 10mM imidazole). Used the standard protocol of purification and elute with 50mM, 300mM imidazole. Flow through fraction have maximum protein and eluted fraction have less protein. I have attached the gel image.
From your gel, the 300 mM elution has significant protein concentration, you just need to run an imidazole gradient.
I would consider checking three things: the protein, the column, and the buffer.
Your statement that the flow-through fraction has the most protein suggests that you think that the band between 63 and 75 kDa is your protein--is that correct? That band runs fast for your expected molecular weight of 80 kDa. The top band in the elution 1 runs closer to what would typically be expected, although it is still unusual for a His-tagged protein to be eluted by only 20 mM imidazole on a nickel column. You can quickly verify which band(s) correspond to your protein if you have a cleavage site for ULP1 or TEV after your affinity tag by cleaving a portion of the sample and running it next to the uncleaved sample on the gel to see which band shifts.
Another thing you might consider doing is regenerating your nickel resin. It is a good idea to do this at least every five uses regardless of whether there are problems. Among other things, the nickel ions in the resin can be reduced by reducing agents in your buffer, causing less binding. This could be one explanation for the fact that the band between 63 and 75 is in all of the fractions.
Alternatively, if your expression level is really high, you could be overloading your column, which is always a nice problem to have. You could divide your sample in half to see if it binds better. This is unusual if the column is in good shape because a 5 mL nickel column can usually bind to more than 100 mg of protein.
There may also be something in your buffer that is preventing binding to Nickel. I am not familiar with how ammonium sulfate affects Nickel resin binding, but you might want to try a dialysis into your Nickel buffer A to see if that improves binding.
Hope something in here is helpful! Good luck!
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